1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-21 to 2011-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is conducted in accordance with GLP regulations and OECD/EU guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted: 27th July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, EPA Health Effects Test Guidelines, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Incorez 397

IUPAC Name:

Incorez 397

Constituent 2

Reference substance name:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

EC Number:

619-764-7

Cas Number:

173904-11-5

Molecular formula:

C16H30N2

IUPAC Name:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

other: Hsd.Brl.Han:Wist

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male animals:9 – 10 weeks old
Female animals:12 – 13 weeks old
- Weight at study initiation:
Male animals:258 – 310 g
Female animals:190 – 223 g
- Fasting period before study:
No data
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: 2 animals / cage

- Diet (e.g. ad libitum):
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water (e.g. ad libitum):
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (Helianthii annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil, at concentrations of 25 mg/mL, 125 mg/mL and 375 mg/mL. Formulations were prepared in the laboratory of Test Facility daily. Analytical control of concentration of dosing solutions was performed in the laboratory of the Test Facility in the second and in the last treatment week (July 04 and August 01, 2011). The measured concentrations ranged from 93.7 % and 109.5 % of nominal concentrations. A report of the formulation analysis was attached in Appendix XV.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water; therefore sunflower oil (Oleum helianthi) was used for preparing formulations appropriate for oral administration. Oleum helianthi /sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle (if gavage):
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.
- Lot/batch no. (if required):
2011.03.04.

Details on mating procedure:

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male until copulation occurred except animal no. 427 which was mated with male animal no. 408 instead of no. 407 by a technical mistake on the last day of mating period.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of Incorez 397 was carried out with a GC method using MS detection. For analysis of the achieved concentrations of Incorez 397 in a formulation the sample was diluted with n-Hexane. The test item was formulated in sunflower oil, at concentrations of 25 mg/mL, 125 mg/mL and 375 mg/mL. Formulations were prepared in the laboratory of Test Facility daily. Analytical control of concentration of dosing solutions was performed in the laboratory of the Test Facility in the second and in the last treatment week (July 04 and August 01, 2011). The measured concentrations ranged from 93.7 % and 109.5 % of nominal concentrations.
The suitability of the chosen vehicle for the test item (stability and homogeneity) was analytically proven. Incorez 397 proved to be stable in sunflower oil formulations at 25 mg/mL and 500 mg/mL concentration levels at room temperature for 24 hours.. A separate analytical report provided these results (Study no. 644.199.2776)

Duration of treatment / exposure:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after 6 days acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 41days (14 days pre-mating and 14 days mating and 13 days post mating), then they were sacrificed on day 41.
Female animals were dosed for 14 days pre-mating, for up to 14 days mating, through gestation and up to lactation days 3, 4 or 5 except two moribund dams which were treated up to and including gestation day 22. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (day 39 and day 40, respectively.
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Frequency of treatment:

Animals were treated once per day.

Details on study schedule:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after 6 days acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 41days (14 days pre-mating and 14 days mating and 13 days post mating), then they were sacrificed on day 41.
Female animals were dosed for 14 days pre-mating, for up to 14 days mating, through gestation and up to lactation days 3, 4 or 5 except two moribund dams which were treated up to and including gestation day 22. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (day 39 and day 40, respectively.
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose setting was based on findings obtained in a previous oral repeated dose toxicity study with Incorez 397 in rats (Study no. 644.120.2464) and in agreement with the Sponsor.
The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Positive control:

NA

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
All parent animals were weighed with an accuracy of 1 g. Parent male animals were weighed on the first day of dosing (day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 20 and on postpartal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals was weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Oestrous cyclicity (parental animals):

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male until copulation occurred except animal no. 427 which was mated with male animal no. 408 instead of no. 407 by a technical mistake on the last day of mating period.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter weight on postnatal days 0 and 4, †
Mean body weight gain per litter between postnatal days 0-4 †
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4 †
* Survival Index of pups on postnatal day 4 †
* Sex ratio % (on postnatal days 0 and 4). †

† = A statistical evaluation was performed
* = Formulas for calculations are given below


GROSS EXAMINATION OF DEAD PUPS:
Pups were carefully examined for gross abnormalities and euthanized on postnatal days 4, 5 or 6.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals:
Male animals were dosed for 41days (14 days pre-mating and 14 days mating and 13 days post mating), then they were sacrificed on day 41.
- Maternal animals:
Female animals were dosed for 14 days pre-mating, for up to 14 days mating, through gestation and up to lactation days 3, 4 or 5 except two moribund dams which were treated up to and including gestation day 22. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (day 39 and day 40, respectively.

GROSS NECROPSY
Gross necropsy was performed on each animal one day after the last treatment. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, colour, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weight of the following organs of parental animals were determined at the necropsy:
With a precision of 0.01 g: uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
With a precision of 0.001 g: ovaries, pituitary

Detailed histological examination was performed on the ovaries, uterus, vagina, pituitary, testes and epididymides of the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
In the cases of moribund animals (nos. 123 and 324), a full histopathology was performed on all sampled organs and tissues as listed in the Study plan.

Postmortem examinations (offspring):

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4, †
Mean body weight gain per litter between postnatal days 0-4 †
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4 †
* Survival Index of pups on postnatal day 4 †
* Sex ratio % (on postnatal days 0 and 4). †

† = A statistical evaluation was performed
* = Formulas for calculations are given below

Statistics:

The statistical evaluation of appropriate data (marked †above) were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.

Reproductive indices:

The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
750 mg/kg bw/day
In the male animals, salivation (12/12), decreased activity (12/12), prone position (12/12), nuzzling up of bedding (12/12) and narrow eye orifice (5/12) were observed at the daily observations after the treatment.
In the female animals, salivation (12/12), decreased activity (12/12), prone position (7/12), nuzzling up of bedding (12/12) and narrow eye orifice (1/12) appeared during the pre-mating period.
In the course of the gestation period clinical observations on salivation (12/12) and decreased activity (12/12) were unchanged, the incidence of activity decrease (5/12) and prone position (3/12) was less than in the pre-mating period and narrow eye orifice was not detected.
In the lactation period, only salivation (12/12) and nuzzling up of bedding (12/12) were observed.

250 mg/kg bw/day
Salivation was noted for all male (12/12) animals in the course of the treatment period.
In female animals, salivation appeared during the pre-mating (12/12), gestation (11/12) and lactation (4/12) periods. Alopecia on the thorax was present in one female rat (1/12) from gestation day 20 up to the necropsy on lactation day 3. The fur was reddish-brown coloured around the left eye of single animal (1/12) from day 10 of the study up on lactation day 3.

50 mg/kg bw/day
There were no clinical signs in the male animals.
In the female group, alopecia was observed in two rats (2/12). In one of them, moderate alopecia on the abdomen was recorded from gestational day 17 up to the lactation day 3. In the other case, alopecia was observed on forelimbs (slight, moderate or marked) from gestational day 3 up to lactation day 3, on the back (slight) from gestational day 3 to 11, and on the right side of the body (moderate or marked) from gestational day 7 up to lactation day 3.

Control group
All male and female animals were symptom-free during the entire observation period.
In summary, test item related clinical signs appeared at 750 mg/kg bw/day (salivation, decreased activity, prone position, nuzzling up of bedding and narrow eye orifice) and at 250 mg/kg bw/day (salivation) in male and female animals after the daily administration. An adaptation to the test item was concluded based on a lower incidence of some signs at 750 mg/kg bw/day during the second half part of the treatment period. Salivation and nuzzling up of bedding were mostly observed both in male and female animals during this time.
Alopecia and reddish-brown coloured fur around the left eye were considered to be individual signs. These occurred only at 50 and 250 mg/kg bw/day and are commonly seen also in untreated Hsd.Brl.Han: Wistar rats.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
750 mg/kg bw/day
The mean body weight of male rats was less than that of the control group from day 7 throughout the entire treatment period. The body weight gain remained below the control value on weeks 1, 2 and 4 resulting in a significantly less total body weight gain (p< 0.01 in each case).
In the female animals, statistically significantly differences were noted for lower body weight gain data during the first week of pre-mating period
(p< 0.01) and for a lower body weight on lactation day 0 (p< 0.01; 1 %). The mean total body weight gain during the premating period remained below the control value (-34 %) but without statistical significance.
The mean body weight gain exceeded the control value during the first week of gestation (p< 0.05).

250 mg/kg bw/day
In the male animals, the mean body weight gain was below the control value between post-mating days 35 and 41.
In the female animals, the mean body weight gain was higher than in the control group during the first week of gestation (p< 0.01).

50 mg/kg bw/day
The mean body weight and body weight gain was similar to the control value in male and female animals during the pre-mating, mating, post-mating and gestation periods. The body weight exceeded the control value on lactation day 0 (p< 0.01; 1 %).
In summary, 750 mg/kg bw/day of Incorez 397 caused a reduction in body weight development in male animals during the entire study while body weight gain of female animals was transiently influenced on week 1 of the premating period. The reduced body weight of male animals, observed at the end of the study, was manifested in the first two weeks as cause of a significantly reduced body weight gain, resulting from decreased food intake during the first two weeks of the study. As the body weight gain went normal in week three, an adaptation to the test item was indicated. However, as histopathology was only performed on reproductive organs and the effects were in line with clinical signs a toxicological relevance could not be excluded.
During the administration of 250 mg/kg bw/day dose, there was no clear evidence of a test item related effect on the body weight development. A slightly lower body weight gain in male animals at the end of treatment period did not resulted in body weight change and was not considered biologically relevant.
Statistically significantly higher body weight gain of female animals with respect to controls at 50 mg/kg bw/day on lactation day 0 was toxicologically not relevant.

FOOD INTAKE (PARENTAL ANIMALS)
750 mg/kg bw/day
The mean daily food consumption was significantly reduced in comparison with the appropriate control value in the male and female animals during the first and second week of the pre-mating period. (p< 0.01; p< 0.05).

250 mg/kg bw/day
In the male animals, the mean daily food intake remained slightly below the control value on the last week of treatment (-7 % between days 35 and 41).
In the female animals, a slightly less food consumption was observed during the entire pre-mating period (weeks 1 and 2; -6 % and -8 %, respectively).

50 mg/kg bw/day
The mean daily food consumption was similar in male and female animals during the pre-mating, post-mating and lactation periods. In the course of last week of gestation period, the mean daily food consumption was higher than in the control group.
In summary, a test item influence on the food consumption of male and female animals was demonstrated at 750 mg/kg bw/day during the first two weeks of the treatment period. At 250 mg/kg bw/day, statistical significances were not considered to be toxicologically relevant as these were of a low degree (< 10 %; male animals during the last week of treatment and female animal during the pre-mating period).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number and percentage of mated and fertile male animals, as well as the copulatory and fertility indices were not affected by the treatment at any dose level. The number and percentage of fertile males, copulatory and fertility indices were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups were similar to the appropriate control values in all test item treated groups. The mean pre-coital interval was similar in the control group and the test item treated groups. Dam with stillborns occurred only in the control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the male animals, statistical significances in the weight of brain, testes and prostate were due to the slightly lower body weight value at 750 mg/kg bw/day.
In the female animals, weight of pituitary was less than in the control at 750 mg/kg bw/day (relative to body weight), at 250 mg/kg bw/day (absolute and relative to body and brain weights) and at 50 mg/kg bw/day (relative to body weight).
In summary, the results of the determination of absolute and relative organ weights did not demonstrate any test item related organ weight alterations. There were no toxicologically significant differences between the control and test item treated groups regarding the examined organ weights. Statistical significances in the weight of brain, testes and prostate were due to the slightly lower body weight value of male animals dosed with 750 mg/kg bw/day. Slight changes in pituitary weight of female animals were not related to doses and were without any supporting histopathological findings. Therefore were not considered toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the control female animal (no. 123) subjected to necropsy in moribund condition bleeding from vagina, piloerection, paleness and cold body temperature were noted at the observation of external appearance. Dead foetuses, foetal death, clotted blood and placenta were found in the uterus,
In moribund animal dosed with 250 mg/kg bw/day (no. 324), piloerection, paleness and bleeding from vagina, in the uterus clotted blood and dead foetuses were detected.

In the male animals, pale kidneys (1/12) were observed at 250 mg/kg bw/day dose.
In the female animals, bilateral pyelectasia (1/12 at 50 mg/kg bw/day), in the uterus early resorption (1/12 at 750 and 1/12 at 50 mg/kg bw/day doses) and late resorption (1/12 at 250 mg/kg bw/day) were observed at the terminal necropsy. Clotted blood was found in vagina of one dam (1/12) dosed with 50 mg/kg bw/day. At the observation of external appearance, alopecia was noted for some dams (2/12 at 50 mg/kg bw/day and 1/12 at 250 mg/kg bw/day).

In summary, no test item related macroscopic alterations were found at the necropsy. Necropsy findings of dead animals (external observations, bleeding from vagina, piloerection, paleness and cold body temperature) were due to the elaborated parturition. Early and late resorptions and clotted blood in vagina occurred with low incidence and were not related to the test item effect. Pale kidneys, pyelectasia and alopecia are species-specific alterations, which commonly occur in animals of this species and strain. These were noted for single animals dosed with 50 mg/kg bw/day or 250 mg/kg bw/day.
All observed effects can be seen occasionally in experimental rats and were not considered as test item related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In moribund animals, histological investigation revealed mild and moderate alveolar emphysema as well as minimal and mild passive hyperaemia in the lungs as a consequence of dyspnoea. In animal no. 324, haemorrhage was seen histologically in the uterus, probably in connection with the parturition. Degenerative, necrotic or any other lesions – in connection with a probably toxic effect – were not detected in the investigated organs of these animals.
In the male animals the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoa and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. Histologically, epididymides, and pituitary gland were normal in all cases as well.
In the female animals including not mated and non-pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.

In summary, histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles with coagulating gland) and pituitary did not reveal any toxic or other test item related alterations at 750 mg/kg/bw/day dose.

OTHER FINDINGS (PARENTAL ANIMALS)
750 mg/kg bw/day
The number of post-implantation loss, total intrauterine mortality, corpora lutea and number and percent of stillborns was less than in the control group and number of live born was higher than in the control group.

250 mg/kg bw/day
The number of corpora lutea was slightly below the control value.

50 mg/kg bw/day
The number of post-implantation loss, total intrauterine mortality, and number and percent of stillborns were lower while the number of live born was higher than in the control group.
In summary, there were no test item related differences between the control and dosed groups in the delivery data of dams. The statistical significance noted for slightly less corpora lutea was not considered biologically relevant as values remained within the historical control range (mean: 14.4; SD: 2.16; n_15; min: 11; max: 18;) moreover pre-implantation loss and total intrauterine mortality were highest in the control group than in 750 mg/kgbw/day group.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs; body weight; food consumption.

2

Dose descriptor:

NOAEL

Remarks:

(reproductive performance)

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There was no test item effect on pups’ mortality.
Extra uterine mortality did not occur in the 750 mg/kg bw/day group. The highest incidence of mortality was detected at 50 mg/kg bw/day (7/114; 6 %). The survival indices were similar between all groups and it was 100 % in the 750 mg/kg bw/day group.

CLINICAL SIGNS (OFFSPRING)
Test item related clinical signs did not appear in pups. The number and percent of pups without findings exceeded the control value at 750, 250 and 50 mg/kg bw/day. The number and percent of cold, not suckled (no milk in the stomach) pups were the highest in the control and 50 mg/kg bw/day groups.

BODY WEIGHT (OFFSPRING)
The mean litter weight and mean pup weight were similar in the control and test item treated groups, no test item related changes were found.

SEXUAL MATURATION (OFFSPRING)
There were no significant differences between control and test item treated groups in the ratio or in the litter means of genders.

GROSS PATHOLOGY (OFFSPRING)
750 mg/kg bw/day
No dead or stillborn pups were found in the high dose group.

250 mg/kg bw/day
Two dams delivered stillborn pups. The lung flotation test was negative in each case indicating that these pups died in the uterus i.e. were stillborn. Autolyzed visceral organs were observed in stillborn pups of dam no. 323 (2/2). In pups of moribund dam no. 324, no macroscopic findings were found (3/3).

50 mg/kg bw/day
Seven live born pups of two dams were necropsised on the day of delivery. The lung flotation test was positive in these pups indicating that pups died after birth. The 6 pups of dam no 222 did not have milk in the stomach (6//6) and pup of dam no. 225 was partially cannibalized (1/1).

Control group
In the control group, 7 dead pups were subjected to necropsy on postnatal day 0. Results of the lung flotation test of the six pups of moribund animal (no. 123) indicated that pups died in the uterus. No macroscopic findings were found in five pups (5/6) and autolysis of visceral organs did not allow further inspection in one pup (1/6). In pup belonging to dam no.125, the performed lung flotation test was positive, indicating that this pup was alive at the birth and died shortly thereafter. There was no milk in its stomach.

In summary, no test item related macroscopic alterations were found in offspring subjected to gross pathological examination. The number of stillborn pups was higher in the control group than in the high dose group.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

(developmental)

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The reprotoxic properties of Incorez 397 were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL was determined to be 250 mg/kg bw/day. The NOAEL for reproductive performance of parental animals was determined to be 750 mg/kg bw/day. The NOAEL for the F1 generation was determined to be 750 mg/kg bw/day.

Executive summary:

The purpose of the reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Incorez 397 on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses.

Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 mg/kg bw/day (vehicle control), 50 mg/kg bw/day, 250 mg/kg bw/day and 750 mg/kg bw/day and at concentrations of 25, 125 and 375 mg/mL corresponding to 2 mL/kg bw dose volume.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations. Incorez 397 proved to be stable in sunflower oil at room temperature for 24 hours. Concentration of the test item in the dosing solutions varied in range of 93.7 % and 109.5 % in comparison to the nominal values at the two analytical occasions in the course of study.

All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout 14 days mating. Test item or vehicle was administered to male animals until 13 days post mating. For females, test item was administered through the gestation period and up to lactation day 3, 4 or 5, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups.

The dams were allowed to litter, and rear their young up to termination on days 4, 5 or 6 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups.

Results

There was no test item related mortality.

Two dams (one control and one dosed with 250 mg/kg bw/day) were euthanized in moribund condition due to an elaborated delivery on lactation day 0.

Clinical signs related to the test item were detected at 750 mg/kg bw/day (salivation, decreased activity, prone position, nuzzling up of bedding and narrow eye orifice) and 250 mg/kg bw/day (salivation) in male and female animals. No signs related to the test item were found at any dose level at the detailed weekly clinical observations indicating animals recovered quickly from signs caused by the daily treatment.

The body weight gain was reduced at 750 mg/kg bw/day during the first two weeks and on week 4, resulting in a lower total body weight in male animals during the entire observation period. A depressed body weight gain was noted for female animals on treatment week 1 then the body weight development was undisturbed in each treatment group during the gestation and lactation periods.

The food consumption was decreased during the first and second week (male and female) at 750 mg/kg bw/day.

There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

Necropsy, organ weight and histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level. Negative effects of the test item on offspring development were not detected.

Conclusion

Under the conditions of the present study, Incorez 397 caused clinical signs, reduction in body weight and food consumption at 750 mg/kg bw/day in male and female Hsd.Brl.Han: Wistar rats after repeated dose oral administration. At 250 mg/kg bw/day, salivation was observed immediately after the administration.

Incorez 397 did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) and development of the F1 offspring from conception to day 4 (occasionally to days 5, 6) post-partum.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for male rats: 250 mg/kg bw/day

NOAEL for female rats: 250 mg/kg bw/day

NOAEL for reproductive performance: 750 mg/kg bw/day